B23 interacts with PES1 and is involved in nucleolar localization of PES1

Acta Biochim Biophys Sin (2009): 991 – 997 | ª The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmp096. Advance Access Publication 12 November 2009 B23 interacts with PES1 and is involved in nucleolar localization of PES1 Jianhua Zhang1, Ying Yang1, and Jiarui Wu1,2 * 1 PES1, the human homolog of zebrafish pescadillo, is a nucleolar protein that is essential for cell proliferation. We report herein that a nucleolar marker protein B23 physically interacts with PES1 and is involved in the nucleolar localization of PES1. In vivo interaction between B23 and PES1 was verified by co-immunoprecipitation of endogenous B23 and PES1 proteins, and they showed cellular co-localizations under both normal and actinomycin D-induced stress conditions. Furthermore, we mapped their interaction domains via in vitro pulldown assays. When B23 was knocked down by RNA interference, there appeared an increased nucleoplasmic distribution of PES1. Our results support a previous hypothesis that B23 might be a nucleolar hub protein for protein targeting to the nucleolus, and shed light on the nucleolar localization mechanism of PES1. The physical interaction between B23 and PES1 implies that they may participate in ribosome biogenesis in a protein complex. Keywords B23; PES1; cell nucleolus Received: July 12, 2009 Accepted: August 18, 2009 Introduction B23 (also known as nucleophosmin, NPM1, or NO38) is an abundant nucleolar protein taking part in various distinct cellular activities including ribosome biogenesis, centrosome duplication, molecular chaperoning and genomic stability control [1–3]. B23 is located primarily in the granular regions of the nucleolus, while many cellular proteins physically interact with B23, such as tumor suppressor ARF [4,5] and P53 [6], ribosomal proteins L5 and S9 [7,8], and interestingly some viral nucleolar targeting proteins [9 –11]. B23 was proposed to be a nucleolar hub protein [12–14]. Notably, B23 is overexpressed in various tumors, and it has been proposed as a marker for gastric, colon, ovarian, and prostate carcinomas. In addition, disruption of the B23 gene by translocation is frequently found in human hematopoietic malignancies. About one-third of adult acute myeloid leukemia (AML) contains B23 mutants with aberrant cytoplasmic distribution [1–3]. Pescadillo, which was originally identified in a mutagenesis screen in zebrafish [15], is highly conserved from yeast to human. Yeast, mouse and human homologs of pescadillo were named as Yph1p, Pes1 and PES1, respectively. Pes1 knockout mice showed embryonic lethality due to a disruption in ribosome biogenesis [16]. Subsequent investigations revealed that PES1 interacted with Bop1 and WDR12, and the three proteins formed a stable complex in the nucleolus involving pre-rRNA processing and maturation of the large ribosomal subunit [17–19]. In addition, pescadillo was reported to regulate gene transcription [20] and induce large-scale chromatin unfolding [21]. Mis-regulation of pescadillo has been associated with cancer and chromosomal instability [22–24]. PES1 is predominantly localized in the nucleolus, with slight distribution in the nucleoplasm [22]. Nucleolar localization is fundamental for PES1 functioning in ribosome biogenesis, since PES1 mutants of domain deletions or highly conserved residue point mutations, which showed diffused nucleoplasmic distribution, failed to replace the function of endogenous PES1 [25]. Despite the importance of nucleolar localization for PES1, what factors maintain PES1 in the nucleolus remain largely unknown. We report herein that the nucleolar marker protein B23 physically interacts with PES1 and is involved in its nucleolar localization. Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 991 Key Laboratory of Systems Biology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 2 Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei 230027, China *Correspondence address. Tel: þ86-21-54921128; Fax: þ86-21-54920787; E-mail: B23 interacts with PES1 Materials and Methods Cell culture, transfection and stable cell line generation HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum (Gibco) at 378C in 5% CO2-containing atmosphere. Cells were transfected with plasmids by jetPEI (Polyplus, Illkirch, France) according to the manufacturer’s instructions. To generate stable HeLa cell line expressing GFP-PES1, HeLa cells were transfected with pEGFP-C1-PES1 and then selected in the presence of 400 mg/ml G418 (Promega, Madison, USA). Immunoprecipitation and immunoblotting For immunoprecipitation, cells were scraped into a lysis buffer containing 50 mM HEPES, pH 7.4, 150 mM Acta Biochim Biophys Sin (2009) | Volume 41 | Issue 12 | Page 992 Protein purification and pull-down assays All GST-PES1 and His-B23 proteins were expressed in E. coli BL21(DE3) cells. Expressions of all GST-PES1 proteins were induced with 0.1 mM IPTG at 288C for 5 h. GST fusion proteins were purified by binding to glutathione-Sepharose 4B (GE Healthcare). Expression of His-B23 full-length and deletion mutants were all induced by 0.1 mM IPTG at 378C for 3 h. Ni-NTA agarose (Qiagen, Hilden, Germany) was used to purify 6 His-tagged B23 proteins. For pull-down assays, HeLa cells were lysed in phosphate-buffered saline (PBS) (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3) plus 1% Triton X-100 and 10 ml/ml proteinase inhibitor cocktail (Sigma), and then centrifuged at 15,300 g for 10 min. The supernatants were collected and incubated with bacterially purified proteins which had been coupled to beads, at 48C for 2 h. Pull-down beads were washed four times with the lysis buffer, and boiled in 2 SDS-loading buffer for 5 min prior to immunoblotting assays. RNA interference and isolation of nucleoli The siRNA targeting B23 was prepared as described previously [26]. The siRNA oligos were transfected into cells by Lipofectamine 2000 (Invitrogen) and maintained for 3 days prior to biological assays. Nucleoli were isolated from HeLa cells as previously described [27], with detailed protocol at http://www.lamondlab.com/ f7nucleolarprotocol.htm. Plasmid construction Full-length PES1 amplified from human cDNA by PCR was cloned into pEGFP-C1 (Clontech, Mountain View, USA) and pGEX-4T-1 (GE Healthcare, Piscataway, USA) generating GFP-PES1 and GST-PES1, respectively. Full-length B23 cloned from human cDNA was inserted into pET-28a (Novagen, Madison, USA) to encode 6 His-tagged B23. Deletion mutants (...truncated)